62 CHAPTER IV ISOLATION AND IDENTIFICATION OF BACTERIA FROM SEPSIS SAMPLES 4.1 INTRODUCTION Infectious diseases remain major cause of mortality in both child and maternal populations. The mortality rate exceeds more than 1 million each year in neonatal and maternal groups, i.e. 10 15% of all maternal and child deaths globally. The huge majority of these deaths occur in low-income countries where the health care systems are not readily available. [349]. In the last two decades, the mortality rate among children under 5 years old has declined considerably; however, neonatal mortality remains unchanged. A predictable 3.1 3.3 million neonates die every year, accounting for 40.3% of underfive mortality. The number of neonates dying in the first month of life per 1,000 live births is estimated around 23.9. In low-middle income countries such as African, Eastern Mediterranean, and Southeast Asia, the neonatal mortality rate ranges from 30.7 35.9, which is substantially greater than high-income countries where it is predictable to be 3.6. Neonatal infections, defined as bacteremia/sepsis, pneumonia, and meningitis, cause around 23.4% of neonatal deaths worldwide. In the last decade, there has been no quantifiable decrease in early neonatal death. To extend research priorities and strategies for deterrence, the mechanisms by which newborns are acquiring bacterial infections require to be better understood [350, 351]. Neonatal sepsis may be classifed as early-onset (EOS) or late-onset (LOS). Of newborns with EOS, 85% present within 24 hours, 5% present at 24-48 hours, and a smaller percentage present within 48-72 hours. Onset is most rapid in premature neonates.
63 Early-onset sepsis is associated with acquisition of microorganisms from the mother. Transplacental infection or an ascending infection from the cervix may be caused by organisms that colonize the mother s genitourinary (GU) tract; the neonate acquires the microorganisms as it passes through the colonized birth canal at delivery. The microorganisms most commonly associated with early-onset infection include the Group B Streptococcus (GBS), Escherichia coli, Coagulase-negative Staphylococcus, Haemophilus influenza and Listeria monocytogenes. Late-onset sepsis occurs at 4-90 days of life and is acquired from the care giving environment. Organisms that have been implicated in causing late-onset sepsis include the following: Coagulase-negative Staphylococcus, Staphylococcus aureus, E coli, Klebsiella, Pseudomonas, Enterobacter, Candida, Group B Streptococci, Serratia and Acinetobacter [25]. Trends in late-onset sepsis show an increase in coagulase-negative streptococcal sepsis; most of these isolates are susceptible to first-generation cephalosporins. [352]. The infant s skin, respiratory tract, conjunctivae, gastrointestinal (GI) tract, and umbilicus may become colonized from the environment, and such colonization to the possibility of late-onset sepsis from invasive microorganisms. Vectors for such colonization may include vascular or urinary catheters, other indwelling lines, or contact with caregivers who have bacterial colonization. Pneumonia is more common in early-onset sepsis, whereas meningitis and bacteremia are more common in late-onset sepsis. Premature and ill infants are more susceptible to sepsis and subtle nonspecific initial presentations; considerable vigilance is therefore required in these patients so that sepsis can be effectively identified and treated. When neonatal sepsis is suspected, treatment should be initiated immediately because of the neonate s relative immunosuppression. Begin antibiotics as soon as diagnostic tests are performed. Blood culture is the current "gold standard" for the detection of blood stream microbial pathogens, it allows microbes to be identified and their susceptibility
64 profiles to be test. The varying microbiological pattern of septicemia in neonates and children warrants the need for ongoing review of the causative organisms and their antimicrobial susceptibility pattern [353]. 4.2 ORGANISMS CAUSING NEONATAL SEPSIS The bacterial profiles for neonatal sepsis in developing countries vary from those seen in developed countries. Gram negative organisms are widely observed and are majorly represented by Klebsiella, E. coli, Pseudomonas, and Salmonella spp. [351, 354]. Of the Gram positive organisms, Staphylococcus aureus [351, 355], Coagulase negative staphylococci (CoNS) [356], Streptococcus pneumoniae (S. pneumoniae) and Streptococcus pyogenes (S. pyogenes) [357, 358] are most frequently isolated. Group B streptococcus is normally rare [359, 360] or not seen at all [361], although similar maternal rectovaginal carriage rates of GBS have been recorded in developed countries [362]. In the majority of the African studies, the occurrence of GBS is low [363, 364], with the exception of South Africa [365]. In Asia, Group B streptococcus is also reported to be very rare [366]. It is not clear whether these differences reflect true variations in pathogens across the world, reflecting an epidemiological evolution in some countries. In developed countries, the neonatal infection study identifies GBS and E coli as the predominant EOS pathogens and CoNS the predominant LOS pathogen followed by GBS and S. aureus [367, 368]. Eerly onset Sepsis disease is often more severe and has higher fatality rate than Late Onset Sepsis. As the LOS is usually caused by CoNS, the associated morbidity and mortality are relatively low [368]. This may not be the case in developing countries where LOS has a higher cause of fatality, particularly Gramnegative bacteria involved.the aim of the present study was to determine the causative organisms for neonatal sepsis among Indian infants, who were cared for, in NICUs in, SRM University India.
65 4.3 MATERIALS AND METHODS 4.3.1 Sample Collection The peripheral blood samples (from Neonatal Intensive Care Unit) and cord blood samples (from Labor Ward) were collected from SRM Hospital, Chennai, India, with the approval of the Institutional Ethical Committee and informed consent was obtained from the parent/guardian of all the subjects enrolled in this study. Cord blood samples were collected within 15 minutes of the delivery by squeezing and puncturing the cord using a clamp. Peripheral blood samples were obtained by vein puncture or drawn from a dwelling arterial or venous line. Both the samples were collected aseptically in collection tubes containing 3.8% sodium citrate as anticoagulant. A total of 68 samples (which included 42 neonates with early onset sepsis and 26 healthy neonates) from both male and female neonates were collected. 4.3.2 Microbial Isolation Sepsis blood samples were collected from infants and they were cultured in Brain Heart Infusion Broth. The culture broth was streaked on a petriplate containing Brain Heart Infusion Agar. After 24 hours, colonies were observed. Individual colonies were then taken and inoculated in Brain Heart Infusion Broth and kept for overnight incubation. The bacterial isolates were subjected to gram staining. Both gram positive and negative bacteria were observed in sepsis samples. Maintenance of bacterial isolates.well grown bacterial colonies were picked and further purified by streaking. The isolated strains were maintained on Nutrient agar slants and stored at 4 C. 4.3.3 Identification of bacteria isolated from sepsis samples Identification of the bacterial isolates was carried out by the routine bacteriological methods i.e., By plating on selective Media s, colony morphology, Gram s staining, Capsule staining, Endospore staining, Motility and biochemical tests.
66 4.3.4 Cultivation of bacteria Nutrient agar is a solid medium that contains nutrient for cultivation of bacteria and fungi. Less than 1% of all existing bacteria can be successfully cultivated, and nutrient agar can grow most of these microbes. Nutrient agar has 66 been used for enumeration of total microorganisms in water, beverages and biological products. Nutrient agar medium was prepared by suspending 20g nutrient agar powder in one litre of deionised water and autoclaving at 121ºC for 15 minutes. The agar was poured into aseptic 9 cm Petri dish and allowed to solidify. The culture was inoculated by an aseptic transfer of 0.1 ml sample aliquot into the medium. After spreading, the medium was allowed to dry and then incubated at 37ºC for 24 hours. Grown cells were enumerated to obtain heterotrophic bacteria counts. 4.3.5 Microscopic characterization 4.3.5.1 Gram staining Gram staining is an pragmatic method used to categorize bacterial species into two large groups Gram-positive and Gram-negative based on the chemical and physical properties of their cell wall. The procedure is based in the ability of microorganisms to retain purple color of the crystal violet during decolorizer with alcohol. A thin smear was prepared on a clean microscopic slide. Heat fixation was carried out showing the slide in flame. The slide was flooded with crystal violet for 1 minute. The slide was rinsed with tap water. A few drops of Gram s iodine was added to cover the smear and allowed to react for 30 seconds. The slide was decolorized with 95% ethanol and rinsed with water. The slide was flooded with safranine and allowed to react for one minute and rinsed with tap water. After decolorization, safranine, a red counter stain, is used to impact a pink color to the decolorizer Gram negative organisms the slide was blot dried and observed under oil immersion objective. Gram negative bacteria are decolorized by the alcohol, losing the purple color of crystal and remain purple.
67 4.3.5.2 Spore staining Spore production is an important characteristic of some bacteria, allowing them to resist adverse environmental conditions such as desiccation, chemical exposure and extreme heat. Malachite green solution was prepared by dissolving 5.0 g malachite green in 100 ml deionised water. Eosin solution was prepared by dissolving 2.5 g eosin Y in 100 ml deionised water and for safranine solution, 0.5 g safranine was dissolved in 100 ml deionised water. The sample for analysis was smeared onto a microscope slide and fixed through drying over a Bunsen burner flame. The slide was placed on a 1000 ml glass beaker and flooded with malachite solution and boiled on a hotplate for 20 seconds. A 30 seconds reaction time was allowed. The slide was rinsed with tap water and then re-stained by flooding it with eosin solution for one minute and then safranine solution for 30 seconds. The slide was rinsed with tap water and softly dried with a paper towel. Positive spore identification was observed as an emerald green color under microscope. 4.3.5.3 Aerobicity and motility tests The motility and aerobicity of bacteria can be determined using a single test medium. Difco manufactures a semi-solid media containing beef extract, peptone, and 0.5% agar. The lower agar content allows bacteria with flagella for movement to demonstrate their motility by moving away from the stab line in an agar deep. Motility can be determined by examining the agar deep. Obligate aerobes require an atmosphere containing concentrations of oxygen similar to room air. Microaerophiles require oxygen, but less than that of room air. Anaerobes do not require oxygen for life, and can be either obligate anaerobes, which grow only in anaerobic environments, or facultative anaerobes, which grow in any oxygen environment. In terms of test results, obligate aerobes appear only at the top of the deep, micro aerophiles grow below the top of the stab line, obligate anaerobes grow 65 only in the bottom portion of the stab line, and facultative aerobes grow evenly throughout the stab and on surface of the culture. Difco bacto-motility test medium (Lot 0105-02) was poured into deeps and allowed to solidify. Deeps were inoculated
68 with a transfer needle and allowed to incubate at 37 C for twenty-four hours before tests were read. 4.3.6 Biochemical Characterization 4.3.6.1 Catalase Test Catalase, an enzyme which breaks down hydrogen peroxide, is frequently found in aerobic and facultative anaerobic bacteria because hydrogen peroxide is a potentially toxic byproduct of aerobic respiration. To test for the presence of catalase, isolated colonies were selected and transferred via a sterile inoculating loop to a clean slide. Hydrogen peroxide (3%, ScholAR Chemistry, Lot AD-5209) was applied drop wise to the bacterial colony on the slide. If the bacteria contained catalase, hydrogen peroxide was converted to water and oxygen gas, causing bubbles to appear on the slide. Catalase negative bacteria produced no reaction. 4.3.6.2 Oxidase test Cytochrome oxidase is the final enzyme in the electron transport chain of bacterial cell respiration. It oxidizes cytochrome C, the electron transport molecule and reduces oxygen to form water. To test for the presence of oxidase, the reduced reagent N N N N -tetramethyl-p-phenylenediamine dihydrochloride (Sigma, Lot 51K1684) was suspended to 1% in deionized water and applied to a smear of the colony under investigation on filter paper. If oxidase was present in the colony, it would oxidize the reagent and the colony on the filter paper would turn dark blue within one minute. A negative oxidase test resulted in no color change of the smear. 4.3.6.3 Lactose utilization The ability of bacteria to utilize lactose as a source of energy and carbon can be tested by the ability of the bacteria to grow on MacConkey agar with salt and crystal violet. Lactose medium selects a wide range of total coliform microbes. Fifty grams of MacConkey agar were weighed into a 2000 ml Erlenmeyer flask, and dissolved in 1000 ml deionised water. The solution was autoclaved at 121ºC for 15
69 minutes. The medium was allowed to cool to about 50ºC, before it was poured into 9 cm aseptic plastic Petri dishes and allowed to solidify. A 0.1 ml subsample of the culture from the sample was aseptically inoculated and spread over the medium with a L-Rod spreader. Inoculated medium was allowed to dry, and then incubated at 37ºC for 48 hours. Microbial cells grown were enumerated. 4.3.6.4 Indole test Peptone broth was prepared and sterilized and it was dispensed into sterile test tubes and culture was inoculated and incubated. After 24 hours, a few drops of Kovac s reagent was added. Formation of red color ring at top of the broth indicates positive result and yellow color indicates negative result. 4.3.6.5 Methyl-red and Voges-Proskauer tests These tests detect the ability of the microorganisms to ferment glucose. For the methyl-red test, glucose is fermented to produce acid. For the Voges-Proskauer test, glucose is fermented to acetoin, and this test enables differentiation of Bacillus 67 species from enterics (International Provisional Standard, 1998). The methyl-red- Voges-Proskauer (MR-VP) broth was prepared by dissolving 17 g of the MR-VP broth in one litre deionised water and dispensing 5 ml aliquots into test tubes. Test tubes containing medium were capped and autoclaved at 121ºC for 15 minutes. For preparation of the methyl-red indicator solution: 0.04 g methyl red was dissolved in 60 ml absolute ethanol and the ph was adjusted to ~5.0. For preparation of O Meara s reagent for the VP test: 40 g potassium hydroxide was dissolved in 100ml deionised water and allowed to cool, then 0.3 g creatine (monohydrate) was dissolved into the reagent. Two test tubes containing MR-VP medium were each inoculated with 1.0 ml of the culture from the same sample and incubated at 35ºC for 4 days. After incubation, the methyl-red test was conducted by adding about five drops of the methyl-red indicator solution to the first tube. A positive result was indicated by the medium changing color to red. The Voges-Proskauer test was conducted by pipetting 5 ml of O Meara s reagent into the second tube. A positive reaction was indicated by the color change to pink within 20 minutes.
70 4.3.6.6 Citrate utilization test Sterile Simmon s citrate agar medium was prepared and poured in test tubes and sterilized. After sterilization, slants were made. The test organisms were streaked with the cultures and incubated at 37 C for 24 hours. After incubation, Color Change from green to blue indicated positive result. No color change indicated negative result. 4.3.6.7 Triple sugar iron agar test TSI agar slants were prepared and test cultures were streaked along the slants and the tubes are incubated at 37 C for 24 hrs. After 24 hrs the tubes are taken and examine the result. 4.3.6.8 Nitrate reduction test Nitrate broth was prepared and dispensed into test tubes. The test tubes were sterilized and one loop full of cultures were inoculated and incubated for 24 hrs. After incubation, few drops of alpha naphthalamine and sulphanilic acid were added. The positive test indicated by red color formation. 4.3.6.9 Sugar fermentation Nutrient broth was prepared with following sugar such as glucose, sucrose, lactose, maltose and mannitol. All these are prepared with indicator (phenol red).the broth was distributed in test tubes and Durham s tube were introduced and sterilized. The organism were inoculate in sugar tubes and incubate the culture for 24-48 hours at 37 C and Observe the results of sugar fermentation have recorded the color changes in broth and gas production, yellow color indicates positive and red color remains means negative.
71 4.3.6.10 Gelatin hydrolysis test Gelatin medium was prepared and plated in a sterile Petri plates. After solidification, the test bacterial cultures were streaked in centre of plate and the inoculated plated were incubated at 37 C for 24 hrs. After incubation, the hydrolyzing activity was tested by using mercuric chloride solution which was flooded on the gelatin agar surface. Formation of clear zone around the line of streak after the addition of mercuric chloride indicates positive result. No clear zone indicates negative result. 4.3.6.11 Starch hydrolysis Some microorganisms contain amylase, an enzyme that can hydrolyse starch into glucose. Amylase is excreted into the media and initiates starch breakdown. The starch hydrolysis test is used to identify the reactions correlated with growth on a starch agar plate [284]. Ten grams of tryptone powder and 15 g of a bacteriological agar were weighed into a 2000 ml Erlenmeyer flask and dissolved in 1000 ml deionised water. The ph of the solution was adjusted to 7.2 using 6 M Hydrochloric acid. The mixture was heated to 95ºC on a hotplate and then 2 g of soluble starch was added and dissolved, then the flask was closed with aluminium foil. The flask was autoclaved at 121ºC for 10 minutes. When the autoclaved medium 69Temperature had decreased to approximately 50ºC, it was poured into 9 cm aseptic Petri dishes and allowed to solidify at room temperature. Samples being analysed were streaked onto starch medium and incubated upside down at 37ºC for 24 hours. Iodine solution was flooded over microbial colonies after the incubation period. In the presence of the enzyme amylase and subsequent starch hydrolysis a yellow/gold zone around the growth was observed and its absence indicated negative results. 4.3.6.12 Urease Test Certain bacteria possess enzymes called ureases which are capable of hydrolyzing urea to yield alkaline ammonia (NH4). The presence of urease is tested
72 for by preparing urea (Sigma, Lot 112F60711) in a broth base containing a ph indicator which is yellow at acidic ph and red at basic ph. Urease liquid media, stored in refrigeration, was poured in three to five milliliter portions into sterile screw-top glass tubes. The tubes were inoculated with the samples in question and allowed to incubate at 37oC for 24 hours, at which point tests were initially read. Pinkish or red tubes were interpreted as positive. Yellow tubes were incubated for another five days at the same temperature. At the end of the five days, if the tubes were pinkish or red they were labeled weakly positive, otherwise they were negative. 4.4 RESULTS AND DISCUSSION Among 42 sepsis samples, the results of the colony morphology, microscopic observation, biochemical test and sugar fermentation test were compared with Bergy s manual (Bergey s manual of determinative bacteriology, 2000, 9 th edition) and the bacterial strains were identified as Staphylococcus aureus 16 (38.09%), Escherichia coli 9 (21.42%), Klebsiella pneumonia 6 (14.28%), Pseudomonas aeruginosa 5 (11.90%), Streptococcus feacalis 3 (7.14%), Acinetobacter antiratus 2 (4.76%) and Klebsiella oxytoca 1 (2.38%) (Table 4.2 and Table 4.3). Table 4.1 Distribution of Gram positive and Gram negative bacteria in sepsis samples. Total number of Samples Sepsis Samples Gram positive samples Gram Negative samples 62 42(61.76%) 19(45.23%) 23(54.76%) Table 4.2 Distribution of isolated bacteria in sepsis samples. S.No. Isolates Number of isolates Proportion 1 Staphylococcus aureus 16 (38.09%) 2 Escherichia coli 9 (21.42%) 3 Klebsiella pneumonia 6 (14.28%) 4 Pseudomonas aeruginosa 5 (11.90%) 5 Streptococcus feacalis 3 (7.14%) 6 Acinetobacter antiratus 2 (4.76%) 7 Klebsiella oxytoca 1 (2.38%)
73 Table 4.3 Biochemical charecteristics of bacteria isolated from sepsis samples. Character S. aureus S.faecalis E. coli Gram Staining Endospore Motility +ve grape-like clusters Nonspore former Non motile +ve, cocci in chains Nonspore former Non motile K.pneu monia -ve rods -ve rods Nonspor e former Motile Nonspo re former Non motile P. aeruginos a -ve cocobacill i Nonspore former Motile K. oxytoc a -ve, Small Rods Nonspo re former Non motile A. antiratu s -ve, Coccoba cilli Nonspor e former Non motile Catalase +ve Negative +ve +ve +ve +ve +ve Oxidase -ve -ve -ve -ve +ve -ve -ve MacConkey Agar Glucose Fermentation Mannitol fermentation Sucrose fermentation Lactose fermentation -ve +ve +ve +ve -ve +ve +ve +ve +ve +ve +ve +ve +ve -ve +ve +ve +ve +ve -ve +ve -ve +ve +ve +ve +ve -ve +ve -ve +ve +ve +ve +ve -ve +ve -ve Indole -ve +ve +ve -ve +ve +ve -ve Methyl redtest -ve -ve +ve -ve -ve -ve -ve VogesProskauer Test Citrate utilization -ve +ve -ve +ve +ve +ve -ve +ve -ve -ve +ve +ve +ve -ve Nitrate reduction +ve +ve +ve +ve +ve +ve -ve Hydrogen sulphide Gelatin hydrolysis Starch hydrolysis -ve -ve -ve -ve -ve -ve -ve -ve +ve -ve -ve +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Urease +ve +ve -ve +ve +ve +ve -ve
74 The epidemiology of neonatal septicemia in the developing and the industrialized countries shows some important differences in the pattern of etiological agents, which often changes over the years. The microbial profile of neonatal septicemia is constantly under change with advances in the early diagnosis and treatment of septicemia. In the pre-antibiotic era, the most common organisms causing septicemia were Gram-positive cocci like Streptococci pyogenes and Pneumococci. With the introduction of antimicrobial agents, Gram-negative organisms like E.coli, Pseudomonas and Klebsiella continues to be a menace to the ill, fragile and debilitated newborns in the neonatal intensive care units [369]. In this study, among the sepsis neonates, 55 % of them were found to be infected by gramnegative organisms such Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter antiratus and Klebsiella oxytoca. For the effective management of neonatal sepsis, knowledge about bacteriological profile play a vital role. In this study, out of 68 samples 42 were culture positive. The incidence of gram positive and gram negative bacteria organisms were 19/42 (45.23%) and 23(54.76%) respectively (Table 4.1). Figure 4.1 Distribution of Gram positive and Gram negative bacteria in sepsis samples.
75 Among gram positive bacteria Staphylococcus aureus were more than all positive isolated organisms and Escherichia coli in gram negative bacteria. Staphylococcus aureus was the predominant pathogen which was isolated from both the early and the late-onset septicaemia cases [370]. In a study done by Sundaram V et al., they reported an increase in the incidence of neonatal sepsis which was caused by S. aureus and a decrease in the incidence of neonatal sepsis which was caused by gram-negative bacilli. Similar findings were obtained in our study [371]. S. aureus was the predominantly isolated gram-positive pathogen in this study; similar findings were evidenced in several studies [351, 372, 373]. Amongst Gram negative organisms, E.coli (21.42%) was found to be the most predominant pathogen, whereas in majority of the studies Klebsiella was found to be the most predominant isolate. The decrease in the incidence of Klebsiella infection in our study could to some extent be explained by the regular monthly fumigation in intensive care unit, rigorous hand washing and drying, the increasing use of disposable syringe and intravenous infusions sets, thus reducing the risk of nosocomial infections [374].The high incidence of E. coli in developing countries may be due to poor hygienic conditions. Gram ve organisms are dominant flora in pregnant females increasing the probability of these organisms gaining access to nurseries and causing infection [375]. In our study E.coli infection is was higher followed by Klebsiella (14.28%), Pseudomonas (11.90%), Acinetobacter antiratus (4.76%) and Klebsiella oxytoca (2.38%). P.aeruginosa is 7% (4/54) and A. baumanii 4% (2/54) similar results were seen in a retrospective study conducted by Mallika Reddy et al., [376]. In developing nations, LOS is complicated by a higher percentage of Gramnegative bacteria and greater antimicrobial resistance among the organisms. Zaidi et al., [377] reported that the rates of neonatal sepsis were 3 20 times higher among hospitalized infants in developing countries compared to developed nations. Klebsiella pneumoniae, other Gram-negative rods (E. coli, Pseudomonas spp., and Acinetobacter spp.), and S. aureus were the major pathogens among 11471 bloodstream isolates [378].
76 A better understanding of this issue would facilitate the necessary behavioral changes in the care of newborn and rational antibiotic therapy in management of neonatal sepsis. More studies are also needed from this area that addresses maternal risk factors for neonatal sepsis in order to provide a better care of the newborn at risk for sepsis [379]. Clinical recognition of neonatal sepsis is not always simple. Suitable intervention requires an early etiological diagnosis. Microbial etiology of neonatal septicemia is diverse. Several studies on neonatal sepsis have recognized the diversity of bacteria and their sequential variability. The present study reiterates the earlier findings and emphasizes the importance of periodic surveys of microbial flora encountered in particular neonatal settings to be familiar with the trend.